The present invention relates to a T cell factor (TCF)-responsive element, a gene and uses of the TCF-responsive element or nucleic acid construct in assays nucleic acid construct comprising a TCF-responsive element and a therapeutic and therapy.
TCFs are a family of transcription factors within the High Mobility Group (HMG) of DNA-binding proteins (Love et al., Nature, 376, 791-795,1995). The family includes TCF-1, TCF-3 and TCF-4 which are described in van der Wetering et al, (EMBO J., 10, 123-132,1991), EP-A-0 939 122 and Korinek et al. (Science, 275, 1784-1787,1997). TCF-4 has been shown to be involved in tumorigenesis related to Wnt/Wingless signalling. TCF and LEF-1 (lymphoid enhancer factor-1) are considered to mediate a nuclear response to Wnt signals by interacting with xcex2-catenin. Wnt signalling and other cellular events that increase the stability of xcex2-catenin are considered to result in transcriptional activation of genes by LEF-1 and TCF proteins in association with xcex2-catenin. In the absence of Wnt signalling, LEF-1/TCF proteins repress transcription in association with Groucho and CBP (CREB binding protein).
In the absence of Wnt signalling, xcex2-catenin is found in two distinct multiprotein complexes. One complex, located at the plasma membrane, couples cadherins (calcium dependent adhesion molecules) with the actin cytoskeleton whereas the other complex (containing the proteins adenomatous polyposis coli protein (APC), axin and glycogen synthase kinase 3xcex2 (GSK3xcex2)) targets xcex2-catenin for degradation. Wnt signalling antagonises the APC-axin-GSK3xcex2 complex, resulting in an increase in the pool of free cytoplasmic xcex2-catenin. The free cytoplasmic xcex2-catenin can translocate to the nucleus where it binds LEF-1/TCF factors and activates Wnt target genes. The regulation of LEF-1/TCF transcription factors by Wnt and other signals is discussed in Eastman et al, (Current Opin. Cell Biology, 11, 233-240, 1999).
The APC gene is a tumour supressor gene that is inactivated in most colorectal cancers. Mutations of APC are considered to cause the accumulation of free xcex2-catenin, which then binds TCF causing increased transcriptional activation of genes including genes important for cell proliferation (e.g. cyclin D1 (Tetsu et al., Nature 398, 422-426, 1999 and Shtutman et al., PNAS USA, 96, 5522-5527, 1999) and c-myc (He et al., Science, 281, 1509-1512,1998)). The involvement of APC in tumour development is discussed in He et al, (supra).
TCFs are known to recognise and bind TCF binding elements which have the nucleotide sequence CTTTGNN, wherein N indicates A or T (van der Wetering et al, supra).
TCF reporter genes have been constructed and are described in Korinek et al, (Science, 275,1784-1787,1997), Morin et al, (Science, 275, 1787-1790, 1997), EP-A-0 939 122 and WO 98/41631. The TCF reporter gene is said to comprise three TCF binding elements upstream of either a minimal c-Fos promoter driving luciferase expression or a minimal herpes virus thymidine kinase promoter driving chloramphenicol acetyl-transferase expression. He et al (supra) discloses TCF reporter gene constructs comprising four TCF binding elements inserted into pBV-Luc.
There is a need for an effective treatment of cancers associated with a deregulation of the Wnt signalling pathway. Such cancers include most colorectal cancers, approximately 30% of melanomas and some breast, prostate and hepatocellular carcinomas.
There is also a need for a TCF response element which when linked to an expressible gene gives improved levels of expression and specificity.
The present invention provides a nucleic acid construct comprising:
a TCF response element comprising:
at least one TCF binding element having the sequence CTTTGNN, wherein N is A or T; and
a promoter,
and an expressible therapeutic gene operably linked to the TCF response element,
wherein the TCF response element enables inducible expression of the operably linked therapeutic gene.
The term xe2x80x9cinducible expressionxe2x80x9d as used herein means the level of expression obtained using the TCF response element is induced (i.e. increased) when one or more TCF/xcex2 catenin heterodimers binds to one or more of the TCF binding elements. Preferably the level of expression is increased by at least 15 fold, more preferably at least 25 fold and most preferably at least 30 fold.
The term xe2x80x9coperably linkedxe2x80x9d as used herein refers to a cis-linkage in which the gene is subject to expression under control of the TCF response element.
The expressible gene comprises the necessary elements enabling gene expression when operably linked to the TCF response element, such as splice acceptor sequences, internal ribosome entry site sequences (IRES) and transcription stop sites. Such elements are well known to those skilled in the art.
It has been found that by using the nucleic acid construct of the present invention that expression of the operably linked therapeutic gene is only induced when TCF/xcex2 catenin heterodimers are present and capable of activating transcription. As cells that have become cancerous due to the deregulation of the Wnt signalling pathway have TCF/xcex2 catenin heterodimers, which activate transcription, expression of the therapeutic gene will be induced. Accordingly, the nucleic acid construct of the present invention acts as a tumour selective promoter.
The nucleic acid construct of the present invention exhibits highly selective expression in that it gives no induction of expression of an operably linked gene above the background level in the absence of TCF/xcex2 catenin heterodimers or a functionally equivalent transcription activating factor.
The therapeutic gene can be any gene that on expression gives a therapeutic benefit. Preferred therapeutic genes include genes encoding toxins such as ricin and diphtheria toxin, and prodrug activating enzymes such as nitroreductases that activate CB1954, cytosine deaminase which activates 5-fluorocytosine, cytochrome P-450 which activates cyclophosphamide and paracetamol, and thymidine kinase which activates ganciclovir. Preferably the therapeutic gene encodes a nitroreductase. Suitable nitroreductases are described in EP-A-0638123 and Watanabe eta/, (NAR, 18, 1059, 1990). Other preferred therapeutic gene include genes encoding immunomodulatory agents such as IL-2, IL-12, GMCSF, B7-1 and B7-2 co-stimulatory molecules; genes encoding tumour suppressers such as RB, p53 and p16; and genes encoding apoptotic genes such as Bax, FasL and caspases.
The promoter can be any promoter that gives a desired level of expression of the operably linked gene. Suitable promoters include the SV40 promoter, the E1B promoter, and the c-Fos promoter. Preferably the promoter is the basal TATA box of the E1B promoter.
Preferably the TCF response element contains at least three and more preferably at least five TCF binding elements. It has been found that by using at least three and more preferably at least five TCF binding elements that an unexpected increase in expression can be obtained compared to a TCF response element containing fewer binding elements. This increase in expression is desirable for the production of a therapeutically effective amount of an encoded product.
Preferably the TCF response element comprises between 5 and 15 TCF binding elements, more preferably between 5 and 10 TCF binding elements and most preferably 5 TCF binding elements.
The TCF binding elements are preferably separated from each other by between 3 and 20 nucleotides, more preferably by between 3 and 14 and most preferably by between 10 and 12 nucleotides.
It is further preferred that the TCF binding elements are so spaced from each other as to be equally distributed radially around the DNA helix, especially when the promoter is the E1B promoter.
It is preferred that the TCF binding element closest to the promoter is between 140 and 10 nucleotides from the TATA box of the promoter. It is further preferred that the TCF binding element closest to the promoter is between 100 and 10 nucleotides, more preferably between 50 and 10 and most preferably between 30 and 15 nucleotides from the TATA box of the promoter.
In one preferred embodiment the TCF binding elements are separated from each other by between 3 or 4 nucleotides and the TCF binding element closest to the promoter is 25 nucleotides from the TATA box of the promoter.
The TCF binding elements preferably have the nucleotide sequence CTTTGAT.
The TCF binding elements can be in either orientation with respect to the promoter, namely 5xe2x80x2 to 3xe2x80x2 or 3xe2x80x2 to 5xe2x80x2.
The present invention also provides a nucleic acid construct designated herein as 5merTCF-E1BTATA, which is shown schematically in FIG. 6 and described in the materials and method section below.
The present invention also provides a TCF response element comprising:
at least five TCF binding elements; and
a promoter sequence,
wherein the TCF response element when operably linked to an expressible gene gives inducible expression of the operably linked gene.
The TCF response element comprising at least five TCF binding elements can be used to obtain inducible expression of any operably linked gene such as a reporter gene or a therapeutic gene. Suitable reporter genes include luciferase, xcex2-galactosidase and chloramphenicol acetyl transferase.
The TCF response element comprising at least five TCF binding elements has been found to give improved (i.e. increased) levels of expression of an operably linked gene compared to a TCF response element comprising less than 5 TCF binding elements.
The TCF binding elements and the promoter of the TCF response element comprising at least 5 TCF binding elements are as defined above.
The present invention also provides a TCF reporter construct comprising the TCF response element having at least 5 TCF binding elements operably linked to a reporter gene.
The present invention also provides the use of the TCF reporter construct of the present invention in a method of identifying candidate drugs for use in the treatment of cancers associated with the deregulation of the Wnt signalling pathway comprising the steps of:
contacting the TCF reporter construct with a test compound under conditions in which the reporter gene is transcribed; and
measuring the transcription of the reporter gene;
wherein a test compound which inhibits transcription of the reporter gene is a candidate drug for cancer treatment.
Preferably the step of contacting the TCF reporter construct is performed in the presence of a lysate from a cell with a deregulated Wnt signalling pathway.
The present invention also provides a vector comprising the nucleic acid construct of the present invention or the TCF responsive element having at least five TCF binding elements of the present invention operably linked to an expressible gene.
The vector may be any vector capable of transferring DNA to a cell. Preferably, the vector is an integrating vector or an episomal vector.
Preferred integrating vectors include recombinant retroviral vectors. A recombinant retroviral vector will include DNA of at least a portion of a retroviral genome which portion is capable of infecting the target cells. The term xe2x80x9cinfectionxe2x80x9d is used to mean the process by which a virus transfers genetic material to its host or target cell. Preferably, the retrovirus used in the construction of a vector of the invention is also rendered replication-defective to remove the effect of viral replication of the target cells. In such cases, the replication-defective viral genome can be packaged by a helper virus in accordance with conventional techniques. Generally, any retrovirus meeting the above criteria of infectiousness and capability of functional gene transfer can be employed in the practice of the invention.
Suitable retroviral vectors include but are not limited to pLJ, pZip, pWe and pEM, well known to those of skill in the art. Suitable packaging virus lines for replication-defective retroviruses include, for example, "psgr"Crip, "psgr"Cre, "psgr"2 and "psgr"Am.
Other vectors useful in the present invention include adenovirus, adeno-associated virus, SV40 virus, vaccinia virus, HSV and poxvirus vectors. A preferred vector is the adenovirus. Adenovirus vectors are well known to those skilled in the art and have been used to deliver genes to numerous cell types, including airway epithelium, skeletal muscle, liver, brain and skin (Hitt, MM, Addison C L and Graham, F L (1997) Human adenovirus vectors for gene transfer into mammalian cells. Advances in Pharmacology, 40: 137-206; and Anderson W F (1998) Human gene therapy. Nature, 392: (6679 Suppl): 25-30).
A further preferred vector is the adeno-associated (AAV) vector. AAV vectors are well known to those skilled in the art and have been used to stably transduce human T-lymphocytes, fibroblasts, nasal polyp, skeletal muscle, brain, erythroid and haematopoietic stem cells for gene therapy applications (Philip et al., 1994, Mol. Cell. Biol., 14,2411-2418; Russell et al., 1994, PNAS USA, 91, 8915-8919; Flotte et al, 1993, PNAS USA, 90, 10613-10617; Walsh et al., 1994, PNAS USA, 89, 7257-7261; Miller et al, 1994, PNAS USA, 91, 10183-10187; Emerson, 1996, Blood, 87, 3082-3088). International Patent Application WO 91/18088 describes specific AAV based vectors.
Preferred episomal vectors include transient non-replicating episomal vectors and self-replicating episomal vectors with functions derived from viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1. Such integrating and episomal vectors are well known to those skilled in the art and are fully described in the body of literature well known to those skilled in the art. In particular, suitable episomal vectors are described in WO98/07876.
Mammalian artificial chromosomes can also be used as vectors in the present invention. The use of mammalian artificial chromosomes is discussed by Calos (1996, TIG, 12, 463-466).
In a preferred embodiment, the vector of the present invention is a plasmid. The plasmid may be is a non-replicating, non-integrating plasmid.
The term xe2x80x9cplasmidxe2x80x9d as used herein refers to any nucleic acid encoding an expressible gene and includes linear or circular nucleic acids and double or single stranded nucleic acids. The nucleic acid can be DNA or RNA and may comprise modified nucleotides or ribonucleotides, and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.
A non-replicating, non-integrating plasmid is a nucleic acid which when transfected into a host cell does not replicate and does not specifically integrate into the host cell""s genome (i.e. does not integrate at high frequencies and does not integrate at specific sites).
Replicating plasmids can be identified using standard assays including the standard replication assay of Ustav et al., EMBO J., 10, 449-457,1991.
The present invention also provides a host cell transfected with the vector of the present invention. The host cell may be any mammalian cell. Preferably the host cell is a rodent or mammalian cell.
Numerous techniques are known and are useful according to the invention for delivering the vectors described herein to cells, including the use of nucleic acid condensing agents, electroporation, complexing with asbestos, polybrene, DEAE cellulose, Dextran, liposomes, cationic liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi, Crit. Rev. Biochem. 16:349-379 (1984); Keown et al., Methods Enzymol. 185:527 (1990)).
A vector of the invention may be delivered to a host cell non-specifically or specifically (i.e., to a designated subset of host cells) via a viral or non-viral means of delivery. Preferred delivery methods of viral origin include viral particle-producing packaging cell lines as transfection recipients for the vector of the present invention into which viral packaging signals have been engineered, such as those of adenovirus, herpes viruses and papovaviruses. Preferred non-viral based gene delivery means and methods may also be used in the invention and include direct naked nucleic acid injection, nucleic acid condensing peptides and non-peptides, cationic liposomes and encapsulation in liposomes.
The direct delivery of vector into tissue has been described and some short-term gene expression has been achieved. Direct delivery of vector into muscle (Wolff et al., Science, 247, 1465-1468,1990) thyroid (Sykes et al., Human Gene Ther., 5, 837-844,1994) melanoma (Vile et al., Cancer Res., 53, 962-967,1993), skin (Hengge et al., Nature Genet, 10, 161-166,1995), liver (Hickman et al., Human Gene Therapy, 5,1477-1483,1994) and after exposure of airway epithelium (Meyer et al., Gene Therapy, 2, 450-460,1995) is clearly described in the prior art.
Various peptides derived from the amino acid sequences of viral envelope proteins have been used in gene transfer when co-administered with polylysine DNA complexes (Plank et al., J. Biol. Chem. 269:12918-12924 (1994));. Trubetskoy et al., Bioconjugate Chem. 3:323-327 (1992); WO 91/17773; WO 92/19287; and Mack et al., Am. J. Med. Sci. 307:138-143 (1994)) suggest that co-condensation of polylysine conjugates with cationic lipids can lead to improvement in gene transfer efficiency. International Patent Application WO 95/02698 discloses the use of viral components to attempt to increase the efficiency of cationic lipid gene transfer.
Nucleic acid condensing agents useful in the invention include spermine, spermine derivatives, histones, cationic peptides, cationic non-peptides such as polyethyleneimine (PEI) and polylysine. xe2x80x98Spermine derivativesxe2x80x99 refers to analogues and derivatives of spermine and include compounds as set forth in International Patent Application WO 93/18759 (published Sep. 30, 1993).
Disulphide bonds have been used to link the peptidic components of a delivery vehicle (Cotten et al., Meth. Enzymol. 217:618-644 (1992)); see also, Trubetskoy et al. (supra).
Delivery vehicles for delivery of DNA constructs to cells are known in the art and include DNA/poly-cation complexes which are specific for a cell surface receptor, as described in, for example, Wu and Wu, J. Biol. Chem. 263:14621 (1988); Wilson et al., J. Biol. Chem. 267:963-967 (1992); and U.S. Pat. No. 5,166,320).
Delivery of a vector according to the invention is contemplated using nucleic acid condensing peptides. Nucleic acid condensing peptides, which are particularly useful for condensing the vector and delivering the vector to a cell, are described in International Patent Application WO 96/41606. Functional groups may be bound to peptides useful for delivery of a vector according to the invention, as described in WO 96/41606. These functional groups may include a ligand that targets a specific cell-type such as a monoclonal antibody, insulin, transferrin, asialoglycoprotein, or a sugar. The ligand thus may target cells in a non-specific manner or in a specific manner that is restricted with respect to cell type.
The functional groups also may comprise a lipid, such as palmitoyl, oleyl, or stearoyl; a neutral hydrophilic polymer such as polyethylene glycol (PEG), or polyvinylpyrrolidine (PVP); a fusogenic peptide such as the HA peptide of influenza virus; or a recombinase or an integrase. The functional group also may comprise an intracellular trafficking protein such as a nuclear localisation sequence (NLS), an endosome escape signal such as a membrane disruptive peptide, or a signal directing a protein directly to the cytoplasm.
The present invention also provides the nucleic acid construct, vector or host cell of the present invention for use in therapy.
Preferably, the nucleic acid construct, vector or host cell is used in the treatment of cancer.
The present invention also provides the use of the nucleic acid construct, vector or host cell of the present invention in the manufacture of a composition for use in the treatment of cancer.
The present invention also provides a method of treatment, comprising administering to a patient in need of such treatment an effective dose of the nucleic acid construct, vector or host cell of the present invention. Preferably, the patient is suffering from cancer.
Preferably, the cancer is any cancer associated with the deregulation of the Wnt signalling pathway such as colorectal cancer, melanomas, breast, prostate and hepatocellular carcinomas.
The present invention also provides a pharmaceutical composition comprising the nucleic acid construct, vector or host cell of the present invention in combination with a pharmaceutically acceptable excipient.
The pharmaceutical compositions of the present invention may comprise the nucleic acid construct, vector or host cell of the present invention, if desired, in admixture with a pharmaceutically acceptable carrier or diluent, for therapy to treat a disease.
The nucleic acid construct, vector or host cell of the invention or the pharmaceutical composition may be administered via a route which includes systemic, intramuscular, subcutaneous, intradermal, intravenous, aerosol, oral (solid or liquid form), topical, ocular, as a suppository, intraperitoneal and/or intrathecal and local direct injection.
The exact dosage regime will, of course, need to be determined by individual clinicians for individual patients and this, in turn, will be controlled by the exact nature of the protein expressed by the therapeutic gene and the type of tissue that is being targeted for treatment.
The dosage also will depend upon the disease indication and the route of administration.
The amount of nucleic acid construct or vector delivered for effective treatment according to the invention will preferably be in the range of between about 50 xcexcg-1000 xcexcg of vector DNA/kg body weight; and more preferably in the range of between about 1-100 xcexcg vector DNA/kg.
Although it is preferred according to the invention to administer the nucleic acid construct, vector or host cell to a mammal for in vivo cell uptake, an ex vivo approach may be utilised whereby cells are removed from an animal, transduced with the nucleic acid construct or vector, and then re-implanted into the animal. The liver, for example, can be accessed by an ex vivo approach by removing hepatocytes from an animal, transducing the hepatocytes in vitro and re-implanting the transduced hepatocytes into the animal (e.g., as described for rabbits by Chowdhury et al., Science 254:1802-1805, 1991, or in humans by Wilson, Hum. Gene Ther. 3:179-222,1992). Such methods also may be effective for delivery to various populations of cells in the circulatory or lymphatic systems, such as erythrocytes, T cells, B cells and haematopoietic stem cells.
The present invention also provides a composition for delivering the nucleic acid construct of the present invention or the TCF response element comprising at least 5 TCF binding elements of the present invention operably linked to an expressible gene to a cell.